Our long term objective is to understand the critical immunopathologic and molecular mechanisms underlying recurrent HSV-1 induced corneal scarring (recurrent herpetic stromal keratitis; R-HSK), a major cause of vision loss in developed countries. Modification of an ocular HSV-1 rabbit model, which closely mimics human R-HSK, resulted in corneal scarring in >70% of eyes (instead of <2%), allowing systematic study of clinically relevant recurrent-HSK for the first time. Preliminary confocal microscopy (CM) studies during latency revealed 2 distinct types of abundant subclinical corneal lesions: 1)EpiLs (epithelial lesions): Transient (~16-24 h), punctate, small (100-200 um diameter), and consistent with replication of reactivated virus from the trigeminal ganglia; 2)SCF (sub-clinical foci): Transient (~3-7 days) clusters of CD45+(RA+/RO+) cells (i.e., immune cells) in the basal epithelium and anterior stroma under a healed EpiL, consistent with the CD45+ cells being attracted to the site by transient viral Ags in the EpiL and then clearing due to lack of continued viral Ag. In corneas without HSK, EpiLs and SCF did not overlap. Occasionally a SCF appeared to evolve into an inflammatory stromal lesion leading to HSK, in which case an overlying EpiL was always seen. We hypothesize that when an active SCF and an active EpiL overlap, viral Ags in the EpiL can restimulate the hyperimmune cells in the SCF resulting in an inflammatory response and HSK. Our Specific Aims include: 1. Using in vivo CM, confirm our hypothesis that epithelial lesions (EpiLs) lead to the formation of transient sub-clinical foci (SCF) and that SCF lead to HSK when a second EpiL occurs at the site of an existing SCF, or when the original EpiL persists until after the SCF is fully formed. The formation and progression (natural history) of EpiLs, SCF, and HSK from acute infection to stromal scarring will be examined daily. We will also microinject viral Ags into the stroma to try to induce HSK. 2. Confirm our hypothesis that SCF are composed of unique subpopulations of primed immune cells responding to viral infection. Identify and characterize immune cell infiltrates and viral Ag in SCF of CJLAT vs wt infected corneas at pre-SCF, SCF, and pre-HSK (EpiL+SCF) times (identified using in vivo CM), by: a) Ex vivo CM; b)In situ proliferation analyses (Ki67 mAb staining/ex vivo CM to identify and quantitate cycling [activated] immune cells); c)Laser MicroDissection (LMD) followed by RNA analysis to detect mRNAs for rabbit cytokines and viral proteins. GFP, YFP, RFP tagged CJLAT and wt viruses will also be employed. 3. Confirm our hypothesis that formation of EpiLs, SCF, and hence, HSK requires replication in the cornea of reactivated HSV-1 returning from latently infected trigeminal ganglia (TG). Changes to HSK and factors Aims 1 and 2 will be re-examined following manipulations of HSK by: a) Antiviral therapy and cutting sensory nerves to/from TG; and b) immuno-suppressive therapy after resolution of the primary infection. The insights obtained should help alleviate this leading cause of infectious corneal blindness. Program Director/Principal Investigator (Last, First, Middle): Wechsler, Steven Lewis Project Narrative This project is aimed at understanding the immunology and molecular biology of herpetic eye disease (HSK), a leading cause of blindness in developed countries.